Compositions for repair of defects in osseous tissues, and methods of making the same

ABSTRACT

Tissue repair compositions, particularly bone repair compositions, containing demineralized bone fragments and homogenized connective tissues, and methods for making the same. The compositions can be used in the form of an injectable gel, an injectable paste, a paste, a putty, or a rehydratable freeze-dried form.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/904,670, filed Feb. 26, 2018, which is a division of U.S. applicationSer. No. 14/689,221, filed Apr. 17, 2015, now U.S. Pat. No. 9,962,467,which is a continuation of U.S. application Ser. No. 13/174,266, filedJun. 30, 2011, now U.S. Pat. No. 9,034,644, which is acontinuation-in-part of U.S. application Ser. No. 12/692,879, filed Jan.25, 2010, which is a division of Ser. No. 10/606,208, filed Jun. 26,2003, now U.S. Pat. No. 7,744,597, which claims priority to U.S.Provisional Application No. 60/391,323, filed Jun. 26, 2002; U.S.application Ser. No. 13/174,266 is a division of U.S. application Ser.No. 11/898,053, filed Sep. 7, 2007, now U.S. Pat. No. 7,977,094, whichis a division of Ser. No. 11/247,229, filed Oct. 12, 2005, now U.S. Pat.No. 7,498,040; all of which are incorporated herein by reference intheir entireties.

BACKGROUND OF THE INVENTION

The ability to promote tissue regrowth in vivo can facilitate woundhealing and post-surgical recovery of patients who have suffered tissuedamage or destruction. A variety of methods and compositions have beenused to repair or regenerate bone tissue in vivo. The need for suchmethods and compositions is readily apparent, when considering that in1999, approximately 500,000 bone graft procedures were performed in theUnited States alone. Ideal bone graft materials for use in suchprocedures possess characteristics necessary to new bone growth, namelyosteoconductivity and osteoinductivity.

Osteoconductivity refers to a graft's ability to support the attachmentof new osteoblasts and osteoprogenitor cells. The osteoconductivecomponents of a graft provide an interconnected structure through whichnew cells can migrate and new blood vessels can form. Osteoinductivityrefers to the ability of a graft to induce nondifferentiated stem cellsor osteoprogenitor cells to differentiate into osteoblasts.

In 1998, 9 of 10 bone graft procedures performed in the United Statesinvolved the use of either autograft or allograft bone tissue. Despitethe benefits of autografts and allografts, the limitations of each havenecessitated the pursuit of alternative graft materials. Using basiccriteria necessary to a successful graft (e.g., osteoconduction andosteoinduction), investigators have developed several bone graftsubstitutes. These can contain a variety of materials, including naturaland synthetic polymers, ceramics, and composites; and in some instances,production of bone graft substitutes can involve biotechnologicalstrategies (i.e., factor- and/or cell-based strategies).

Osteoinductive substances found in some bone graft substitutes aredemineralized bone particles and/or powder. Contained in theextracellular matrix of bone tissue is a full cocktail of bone growthfactors, proteins, and other bioactive materials necessary forosteoinduction and, ultimately, successful bone healing. To capitalizeon this cocktail of proteins, bone tissue can be demineralized, leavingthe osteoinductive agents in the demineralized bone matrix (DBM). Suchosteoinductive DBM can be incorporated into a number of different bonegraft substitutes.

While a number of different materials thought to enhanceosteoconductivity (i.e., purified or partially purified polymers) havebeen used in DBM bone graft substitutes; new, more easily prepared,osteoconductivestructural materials for combining with DBM to produce abone graft substitute are desirable.

SUMMARY OF THE INVENTION

Certain embodiments of the present invention are directed to tissuerepair compositions and methods of preparing the same. Some tissuerepair compositions of the present invention may be in the form of afluid injectable gel, a fluid injectable paste, a putty, or arehydratable freeze-dried paste. Some embodiments of the presentinvention may be used in clinical applications, such as spinalprocedures, orthopedic procedures and dental procedures.

Some embodiments of the present invention are directed to tissue repaircompositions having a plurality of demineralized bone fragments and acarrier. The carrier comprises homogenized connective tissue or skin.The demineralized bone fragments may be demineralized bone particles ordemineralized bone fibers, in certain embodiments. In some embodiments,demineralized bone fragments may be derived from allogenic cortical boneor xenogenic cortical bone. Demineralized bone fragments may, in certainembodiments, have less than about 8 wt % residual calcium. The tissuerepair composition may comprise between about 5 wt % and about 90 wt %demineralized bone fragments, in some embodiments. In certainembodiments, the connective tissue that has been homogenized may befascia, tendon, ligament, pericardium, articular cartilage, or mixturesthereof. Homogenized connective tissue or skin used in some embodimentsof the present invention may be comprised of tissue fragments having anaverage diameter of less than about 50 microns.

Certain embodiments are directed to methods of preparing a tissue repaircomposition. The methods comprise combining a plurality of demineralizedbone fragments and a carrier having homogenized connective tissue orskin. Certain methods further comprise at least one of a bonefragmentation step, a connective tissue or skin fragmentation step, abone demineralization step, a selecting of demineralized bone fragmentsof a particular size range step, a freeze-drying step, a rehydratingstep, a heating step, a connective tissue homogenization step, apackaging step, and a sterilization step.

Certain embodiments of the present invention are directed to prostheticdevices comprising, an implantable prosthetic device, and a coatingdirectly adjacent to at least a portion of a surface of the implantableprosthetic device. The coating comprises at least one tissue repaircomposition comprising a plurality of demineralized bone fragments and ahomogenized connective tissue.

Some embodiments of the present invention are directed to a method ofcoating a prosthetic device comprising, providing an implantableprosthetic device, and applying at least one tissue repair compositionto at least a portion of a surface of the implantable prosthetic device.The tissue repair composition is as described above.

DETAILED DESCRIPTION

In the present application, the term “connective tissue” refers tomesodermally derived tissue that may be more or less specialized, andthat is, at least in part, made up of fibers. Most of the connectivetissues, contemplated in the present invention are less specializedtissues that are rich in extracellular matrix (i.e., collagen,proteoglycan, among others), and that surround other more highly orderedtissues and organs. A relatively, more specialized tissue contemplatedin the present invention is cartilage. Varieties of connective tissuethat may be used in the present invention include: loose; adipose;dense, regular or irregular, white fibrous; elastic; and cartilage.Connective tissue may be classified according to concentration of fibersas loose (areolar) and dense, the latter having more abundant fibersthan the former. Connective tissues may be obtained from vertebrates. Insome embodiments, the tissues may have human, bovine, equine, porcine,ovine, caprine, or piscene origins, among others. Connective tissues mayalso be the product of biotechnological methods (i.e., production oftissue engineered connective tissues using cell culture methods).

Specific examples of connective tissues that may be used in certainembodiments of the present invention include, at least, fascia, tendons,ligaments, pericardium, and articular cartilage. Different types offascia that may be used in certain embodiments of the present inventioninclude: fascia lata, fascia adherens, fascia brachii, fascia axillaris,antebrachial fascia, abdominal fascia, internal fascia, fascia iliaca,fascia profunda, clavipectoral fascia, fascia cribosa, crucial fascia,deltoid fascia, dorsal deep fascia, pelvic fascia, fascia cruris, lumbarfascia, and pectoral fascia, among others. “Crudely fragmentedconnective tissue” or “crudely fragmented skin” refers to connectivetissue or skin, respectively, that has been sliced, ground, carved,chipped, chopped, minced, cut, dissected, rent, ripped, sectioned,snipped, diced, shaved, comminuted, or trimmed into fragments having anaverage diameter greater than about 50 microns and less than about 0.5cm (i.e., having cut dimensions of approximately 0.5×0.5 cm), andthickness appropriate to the tissue being crudely fragmented. In someembodiments, the crude fragments may not be of uniform size.

“Homogenized connective tissue or skin” or “connective tissue or skinhomogenate” contains connective tissue or skin that has been reduced toparticles that are uniformly small and evenly distributed. Homogenizedconnective tissue or skin may optionally include at least one of water,aqueous solutions, or water miscible polar organic solvents, in additionto the particles. The homogenized connective tissues or skin used inmethods of the present invention include particles having an averagediameter of less than about 50 microns. In some embodiments, thehomogenized connective tissue or skin may be prepared by shear-inducedshredding of a composition comprising connective tissue, and optionally,at least one of water, an aqueous solution and a water miscible polarorganic solvent. A conventional blender may be used in preparing thehomogenized connective tissue, in certain embodiments.

“Osseous tissue” is meant to refer to bone tissue, tissue resemblingbone; and tissue capable of forming bone. The term “bone” or “bonetissue” is intended for the purposes of the invention to refer toautograft bone, allograft bone, and xenograft bone. Such bone includesany bone from any source, including: bone from a living human donor,bone from a human cadaveric donor, and bone from an animal. The bone mayinclude cortical bone and/or cancellous bone and/or cortico-cancellousbone. The term “bone fragment,” as used in the present applicationrefers to ground bone, pulverized bone, bone cubes, bone chips, bonestrips, bone particles, and bone fibers. Bone fragments may be “boneparticles” or “bone fibers,” in some embodiments of the presentinvention. “Bone particle” refers to a piece of bone having an averagediameter of between about 125 microns and about 4 mm. “Bone fiber”refers to a filament or thread of bone having an average thickness ofbetween about 0.1 mm and about 1.4 mm and an average width of betweenabout 0.3 mm and about 2.5 mm. Fibers can be of varying lengths. Incertain embodiments, a bone fiber can have an average length of betweenabout 1.0 mm and about 100 mm. In certain embodiments, bone fibercontains lamellae in the shape of threads or filaments having a medianlength to median thickness ratio of about 10:1.

“Demineralized bone,” as used in the present application refers to bonehaving less than about 8 wt % residual calcium. Demineralizationinvolves treating the surface of a bone tissue to remove a surface layerof its inorganic mineral hydroxyapatite material leaving the mechanicalproperties of the organic phase of the bone constructs substantiallyunchanged. The level of demineralization of a bone tissue is defined bythe amount (wt %) of residual calcium found in the demineralized bone.In some embodiments, the demineralized bone may contain physiologicallyactive levels of growth and differentiation factors (i.e., bonemorphogenetic proteins (BMPs)).

In the present application, the term “gel” refers to a jelly-like,thick, soft, partly liquid substance. A gel of the present invention maybe extruded through at least a 13 gauge syringe needle. “Paste,” as usedin the present application refers to a soft, moist, substance having aconsistency between a liquid and a solid. A paste of the presentinvention is less solid than a putty and More solid that a gel, and insome embodiments may be injectable.

“Putty” refers to a dough-like/clay-like tissue repair composition ofthe present invention. During application the substance may be beaten orkneaded to the consistency of dough, and molded into a shape closelyapproximating that of the implant site.

“Injectable” refers to the ability of certain tissue repair compositionsof the present invention to be introduced at an implant site underpressure (as by introduction using a syringe). An injectable compositionof the present invention may, for example, be introduced betweenelements or into a confined space in vivo (i.e., between pieces of boneor into the interface between a prosthetic device and bone, amongothers).

“Syringe” refers to any device that may be used to inject or withdrawflowable tissue repair compositions of the present invention, includingcertain gels and pastes, among others.

“Flowable” refers to the characteristic of a composition that permits itto be made to fit closely by following the contours of a site. Flowablecompositions may be fluid, malleable, plastic, and/or pliable.

“Allogenic tissue” refers to a tissue from a donor that is implantedinto a recipient of the same species. Allograft tissue is widely used inorthopedic, neuro-, maxillofacial, podiatric, and dental surgery. Thetissue is valuable because it is strong, biointegrates in time with therecipient patient's tissue and may be shaped to fit the specificsurgical defect. Contrasted to most synthetic absorbable ornonabsorbable polymers or metals, allograft tissue is biocompatible andintegrates with the surrounding tissues. Allograft bone occurs in twobasic forms: cancellous and cortical.

“Xenogenic tissue” refers to a tissue from one species that is implantedinto a recipient of another species.

“Cortical bone,” as used in the present application, refers to thecompact bone of the shaft of a bone that surrounds the medullary cavity.Cortical bone is a highly dense structure made up of triple helixstrands of collagen fiber, reinforced with hydroxyapatite. The corticalbone is a compound structure and is the primary load bearing componentof long bones in the human body. The hydroxyapatite component isresponsible for the high compressive strength of the bone while thecollagen fiber component contributes in part to torsional and tensilestrength.

Trabecular bone is of similar composition to cortical bone and is theprimary structural component of “cancellous bone” and refers to adultbone consisting of mineralized regularly ordered parallel collagenfibers organized differently than in the lamellar bone of the shaft ofadult long bones. Cancellous bone is generally found in the end of longbones surrounded by cortical bone. Cancellous bone has spicules thatform a latticework, with interstices filled with bone marrow. It mayalso be referred to as a trabecular bone, or spongy bone.

“Aseptic” as a term can be applied to both products and processes and isgenerally applied to the control or reduction in microbial bioburden.Tissues processed “aseptically” are tissues processed using sterileinstruments, and special environmental surroundings (including forexample “clean room technologies”). Aseptic tissues make reference totissues that are “culture negative,” where culture negative makesreference to the use of representative pieces of tissue that have beenor will be processed for an assessment of the presence ofmicroorganisms. The level of sensitivity of the microbiological testmethod(s), and hence a better definition of “culture negative,” isgenerally predetermined by assessing for interference in the detectionof such microorganisms (sometimes referred to as bacteriostasis andfungistasis, B&F, testing).

“Sterile” makes reference to a definition such as contained in the Codeof Federal Regulations (21 CFR.) where the probability of a culturablemicroorganism being present on a processed sample is equal to or lessthan 1 in one million, i.e., a Sterility Assurance Level, or SAL, of1×10⁻⁶.

An “osseous defect” is generally defined by one skilled in the art asbeing an imperfection or void in an osseous tissue, which is ofsufficient physical dimensions as to not heal spontaneously. Hence, theuse of materials generally referred to as “bone void fillers”areutilized clinically to aid or improve healing of the osseous defect.Certain compositions of the present invention can be used as bone voidfillers. Osseous defects may include: fractures, cracks, andosteosarcomas (bone cancer lesions), among others. Bone void fillers maybe used to fill a gap between a prosthetic device and bone; betweenpieces of bone; and between two different prosthetic devices. Forexample, a bone void filler can be used to fill the space between a hipreplacement and a bore in a bone into which the hip replacement has beeninserted

Certain embodiments of the present invention are directed to tissuerepair compositions comprising a plurality of demineralized bonefragments and a carrier. The carrier includes at least one homogenizedconnective tissue. In some embodiments, the tissue repair compositionmay be safely used in repairing damaged osseous tissues (e.g., damagedbone) in an implant patient. The tissue repair composition may, in someembodiments, be biocompatible, osteoinductive, and/or osteoconductive,such that it may ultimately be remodeled to a mineralized, hard tissueat the application site in vivo. In certain embodiments, the tissuerepair compositions may further include at least one of water, anaqueous solution, a water miscible polar organic solvent, and othercomponents described below. The tissue repair composition may includematerials that improve handling or functional characteristicspost-implantation. In some embodiments, bone and connective tissue maybe obtained from the same donor source (i.e., a single human cadaverdonor). The formulation of the inventive tissue repair composition maybe highly reproducible. In certain embodiments, the tissue repaircomposition may be aseptic or sterile.

The composition may be in the form of a gel, a paste, a putty, or afreeze-dried substance that can be rehydrated to produce a paste or aputty. In some embodiments, the gel or paste may be injectable, and thegel or paste may be extrudable through a syringe and/or a syringe havingat least a 13 gauge tube/needle coupled thereto. Certain gels and pastesmay be used for accurate delivery of the tissue repair composition intonarrow junctions with minimal surgical damage to surrounding tissue atthe implant site. Some of the tissue repair compositions of the presentinvention may be moldable. Tissue repair compositions of the presentinvention may be cast into a shaped form, in certain embodiments.

In some embodiments, each of (a) the demineralized bone fragments and(b) the homogenized connective tissue of the inventive composition mayinclude materials derived from allogenic or xenogenic sources. Incertain embodiments, bone and connective tissues obtained fromvertebrate species, for example human, bovine, porcine, ovine, caprine,and piscene sources may be used to prepare demineralized bone fragmentsand carrier. The plurality of demineralized bone fragments may includemore than one type of bone tissue (e.g., cancellous, cortical, orcortico-cancellous bone), and the homogenized connective tissue mayinclude more than one type of connective tissue (i.e., fascia andtendon). The plurality of demineralized bone fragments may include bonefrom a single donor source, or from multiple donor sources, and thehomogenized connective tissue may also include tissue from a singledonor source, or from multiple donor sources.

For preparation of the tissue repair compositions of the presentinvention, the carrier comprising the connective tissue homogenate andthe demineralized bone fragments are combined together. In someembodiments, the tissue repair composition may include between about 5wt % and about 90 wt %; between about 20 wt % and about 80 wt %; andbetween about 30 wt % and about 50 wt % demineralized bone fragments.Certain tissue compositions of the present invention that are in theform of a gel or paste may include between about 20 wt % and about 30 wt% demineralized bone fragments, while certain compositions of thepresent invention that are in the form of a putty may include betweenabout 25 wt % and about 40 wt % of the demineralized bone fragments. Insome embodiments, the tissue repair composition may be in the form of afreeze-dried product that may be rehydrated to produce a paste or aputty, which may include between about 35 wt % and about 50 wt %demineralized bone fragments.

In certain embodiments, the tissue repair composition may includedemineralized bone fragments having less than about 8 wt % or less thanabout 4 wt % residual calcium. The tissue repair composition may includedemineralized bone fragments having between about 0.5 wt % and about 4wt % residual calcium, in some embodiments. In certain embodiments, thetissue repakcomposition may include between about 0.25 wt % and about 80wt % or about 0.5 wt % and about 5 wt % of the connective tissuehomogenate. The amount of homogenized connective tissue used in a tissuerepair composition may be used to adjust the viscosity and gelationcharacteristics of the composition.

The plurality of demineralized bone fragments may include at least oneof demineralized bone particles and demineralized bone fibers, in someembodiments. The demineralized bone fragments may include materialsderived from allogenic or xenogenic sources. The demineralized bonefragments may be derived from cortical bone or cancellous bone. Incertain embodiments, the plurality of demineralizedbone fragmentsincludes at least one of demineralized allogenic cortical boneparticles, demineralized xenogenic cortical bone particles,demineralized allogenic cancellous bone particles, and demineralizedxenogenic cancellous bone particles.

Certain tissue repair compositions of the present invention may includedemineralized bone particles. Demineralized bone particles may beprepared from cleaned and disinfected bone fragments that have beenfreeze-dried and ground/fractured into bone particles. Bone particlesmay be selected by, for example, using sieving deviees (i.e., meshsieves) commercially available to obtain particles within a desired sizerange. Such demineralized bone particles may have an average diameter ofbetween about 125 microns and about 4 mm; between about 710 microns andabout 2 mm; between about 125 microns and about 500 microns; betweenabout 125 microns and about 850 microns; or between about 250 micronsand about 710 microns. Certain embodiments of the present invention mayinclude demineralized bone powder that is commercially available. Forexample, a suitable demineralized bone powder that is widely andreliably available is produced by LifeNet, Virginia Beach, Va.

Some tissue repair compositions of the present invention may includedemineralized bone fibers. Fiber bone may be produced as described inU.S. patent application Ser. No. 10/606,208, published as publicationnumber 2004/0059364, which is hereby incorporated by reference in itsentirety. In certain embodiments, the demineralized bone fibers may havean average thickness of between about 0.1 mm and about 0.3 mm and anaverage width of between about 0.3 mm and about 1.0 mm. The length ofthe fibers may vary. Any demineralization processes known in the art,may be used to prepare demineralized bone fragments. Such processes aredescribed in U.S. Pat. Nos. 6,830,763; 6,534,095; 6,305,379; 6,189,537;5,531,791; and 5,275,954. In some embodiments, the demineralizationprocess begins by producing bone particles having an average diametersize range of between about 1 mm and about 2 mm or bone fibers having anaverage dimension of 0.1 mm to 0.3 mm thick and an average width ofabout 0.3 mm to about 1 mm. The fragments may then be treated by suchprocesses as are described in U.S. Pat. Nos. 5,556,379; 5,797,871;5,820,581; 5,976,104; 5,977,034; 5,977,432; and 6,024,735, which arehereby incorporated by reference in their entirety. If the bone to beprocessed into fragments has not been previously cleaned anddisinfected, they may be cleaned and disinfected by the use ofdetergents, hydrogen peroxides, antibiotics, and alcohols to affect aremoval of associated tissues such as bone marrow and cellular elements.Following a cleaning and disinfection, these fragments (i.e., particlesand fibers) may be demineralized by exposure to dilute hydrochloricacid, such as are known in the art, to affect a removal/reduction of themineral component of the bone fragments (i.e., particles and fibers).Such additional processing may, in some instances, inactivate potentialviral contamination (i.e., HIV and hepatitis viruses, among others).

In certain embodiments in which demineralized bone fragments are to beused later, they may be conveniently stored by freeze-drying, which maymaintain the activity of their bioactive components (i.e., BMPs, amongothers). If the demineralized bone fragments are to be used later, insome embodiments, the acidic demineralization solution may be removedfrom the bone using aqueous or polar (miscible with water) organicsolutions, for example deionized/distilled endotoxin-free water, salinesolutions, acetone, alcohol(s), and dimethylsulfoxide, in order toMinimize elevated levels of salts in the freeze-dried bone.

Tissue repair compositions of the present invention include a carrierhaving a homogenized connective tissue. In some embodiments, the carrierand/or connective tissue homogenate may include a biocompatibleliquefied form of connective tissue (i.e., liquefied human connectivetissue) that when combined with demineralized bone fragments, hassuitable viscosity so as to be injectable through large gaugeapplicators, while largely remaining at the implant site. The carriermay promote cellular infiltration and retain the demineralized bonefragments at the site of application, without being cytotoxic. Thecarrier may promote such cellular infiltration by providing a molecularmatrix for cell migration. In some embodiments, the carrier/connectivetissue homogenate may be freeze-dried.

In some embodiments, the homogenized connective tissue or skin may beprepared from allogenic or xenogenic tissue. Such connective tissue orskin may be obtained from a human donor or an animal (i.e., bovinedonor, porcine donor, etc.). Connective tissue may be obtainedrelatively economically. Varieties of connective tissue that may be usedin certain embodiments of the present invention include: areolar orloose; adipose; dense, regular or irregular; white fibrous; elastic; andcartilage. Specific examples of connective tissues that may be used incertain embodiments of the present invention include, at least: fascia,tendons, ligaments, pericardium, and articular cartilage. Differenttypes of fascia that may be used in some embodiments of the presentinvention include: fascia lata, fascia adherens, fascia brachii, fasciaaxillaris, antebrachial fascia, abdominal fascia, internal fascia,fascia iliaca, fascia profunda, clavipectoral fascia, fascia cribosa,crucial fascia, deltoid fascia, dorsal deep fascia, pelvic fascia,fascia cruris, lumbar fascia, and pectoral fascia, among others. Forpractical reasons of availability during procurement and amount offascia available, fascia lata from the anterior portion of the upper legmay be used in certain embodiments. Homogenized connective tissue orskin may be prepared by methods involving, cleaning and disinfectingconnective tissue or skin, and removing extraneous tissues associatedwith the connective tissue or skin. Connective tissues or skin may becut into small pieces to produce crudely fragmented connective tissue orskin, and optionally triturated and washed with distilled/deionizedendotoxin-free water and/or an aqueous solution (i.e., isotonic saline,among others). In processing, multiple “washes” may be affected usingvolumes of aqueous solution that are 10 times the approximated volume ofthe tissue being processed, in some embodiments. It would be obvious toone skilled in the art that the use of three such processing steps wouldaffect an approximate 1:1000 dilution of associated solubilizableelements rendering the tissue essentially free from such solubilizableelements, Connective tissue or skin may be treated and homogenized attemperatures sufficient to produce a flowable homogenized connectivetissue or skin, in certain embodiments. The homogenized connectivetissue or skin may include connective tissue or skin that has beenreduced to particles that are uniformly small and evenly distributed.Homogenized connective tissue, skin, and/or the carrier may optionallyinclude at least one of water, aqueous solutions (i.e., isotonicsaline), and water miscible polar organic solvents in addition to theconnective tissue or skin particles. In some aspects, the homogenizedconnective tissue or skin may include gelatin. The homogenizedconnective tissues or skin used in methods of the present invention mayinclude particles having an average diameter of less than about 50microns, less than about 20 microns, or less than about 50 microns andgreater than about 5 microns. In some embodiments, the homogenizedconnective tissue, skin and optionally, at least one of a water misciblepolar organic solvent, water and an aqueous solution, may be prepared byshear-induced shredding of connective tissue or skin. A conventionalblender may be used in preparing the homogenized connective tissue orskin, in certain embodiments.

In some embodiments of the present invention, connective tissuehomogenate and/or the carrier will retain large and small molecularweight macromolecules, including hyaluronate which is known to play arole in cell migration (Toole, B. P. and Trelstad, R. L. 1971, Develop.Biol. 26:28-35; Docherty, R. et al., 1989, J. Cell. Sci. 92:263-270) andhas been implicated in facilitating fibril formation which promotesgelation (Tsunenaga, M. et al., 1992. Connect. Tiss. Res. 28:113-123).The connective tissue homogenate and/or carrier may have excellenthistocompatibility and elicit minimal antibody formation orimmunological rejection, in certain embodiments. Keeping this in mind,the homogenized connective tissue may be made acellular, using methodsknown in the art, prior to homogenization, and methods of making suchtissues acellular are described in U.S. Pat. Nos. 6,734,018 and6,743,574; which are hereby incorporated by reference in their entirety.

An acellularization process used to prepare homogenized connectivetissue of the present invention may be performed without damage tomatrix and/or tissue structure, in some embodiments. Mechanical strengthof a connective tissue may reside in the matrix structure of the tissue.The matrix structure may include collagens, hyaluronins, elastins,mucopolysaccharides and proteoglycans, among other components. Anexample of an acellularization method for use with soft tissues isdescribed in U.S. Pat. Nos. 6,734,018 and 6,743,574, which are herebyincorporated by reference in their entirety. Connective tissue that isaceilularized may have a thickness that does not exceed about 8 mm,about 6 mm, or about 4 mm, in certain embodiments. Acellularizationprocessing may be altered to accommodate the thicker tissues.

Certain tissue repair compositions of the present invention may includeelements in addition to the plurality of bone fragments and the carrier.Additional elements may be bioactive compounds, antibiotics (i.e.,penicillin), antiviral agents (i.e., Triton X-100, Nonidet P-40,N-lauroyl sarcosinate, Brij-35, and peroxide generating agents),antitumor agents, analgesics, immunosuppressive agents (i.e., bovineintestinal alkaline phosphatase), permeation enhancers (i.e., fatty acidesters, such as the laurate, myristate and stearate monoesters ofpolyethylene glycol), nucleic acids, mesenchymal elements, gelationenhancing compounds (i.e., hyaluronic acid, chondroitin sulfate,dermatin sulfate, carboxymethylcellulose, methylcellulose, polyethyleneglycol, or glycosamino glycans), or autogenously derived osteoblastcells, among others. Examples of bioactive compounds include: bonemorphogenic proteins, transforming growth factor beta, fibroblast growthfactor, insulin, vascular endothelial growth factor, and plateletderived growth factor, among others. In this respect, the inventionincludes other equivalent optional components readily known to those inthe art. Tissue repair compositions of the present invention may includea calcium phosphate and/or calcium, sulfate mineral component to producean osteoinductive/osteoconductive composition which will harden prior toor post implantation. Tissue repair compositions of the presentinvention may also include particulate hydroxyapatite, calciumphosphate, magnesium phosphate, calcium carbonate, as extenders of thecompositions and as sources of mineral in subsequent induced new boneformation.

In some embodiments, the tissue repair composition or components of atissue repair composition, and optionally means for applying a tissuerepair composition (i.e., syringe or spatula) to an implant site may beprovided in a unitary kit, In other embodiments, the demineralized bonefragments and the connective tissue homogenate and/or the carrier may beprepared under sterile conditions and stored separately, or mixed andstored together, for later use. To facilitate clinical usage ofdescribed tissue repair compositions, the demineralized bone fragmentsand the carrier/connective tissue homogenate may be packaged separatelyin different forms and reconstituted and combined at the time of usage,in some embodiments. In other embodiments, the components may becombined to produce a tissue repair composition, which is then packaged,in a premixed format.

The premixed format provides the advantage of requiring minimalpreparation by the individual clinician at the time of usage. In someembodiments, the tissue repair composition may be stored in anapplication means, such as a syringe, which will be used to apply thecomposition to an osseous defect site. The tissue repair compositionmay, for example, be stored in a 1 to 10 cc syringe that is capable ofbeing coupled to a large gauge delivery tube/needle of appropriatelength and inside diameter. In this regard, a delivery tube with aninside diameter of not less than 13 gauge is appropriate for theinjection delivery into an implant site.

For on-site preparation, the carrier/homogenized connective tissue anddemineralized bone may be provided in freeze-dried aliquots that arerehydrated just prior to being combined for use in clinicalapplications, in some embodiments. On-site preparation has the advantageof increasing the ability to vary the concentrations and quantities ofthe carrier/connective tissue homogenate and demineralized bonefragments used in preparation of the inventive tissue repaircomposition. Furthermore, on-site preparation permits the addition ofoptional components at the discretion of the clinician.

Certain embodiments of the present invention are directed to methods forthe preparation of inventive tissue repair compositions, as describedabove. Such methods include combining a plurality of demineralized bonefragments and a carrier comprising a homogenized connective tissue.Certain inventive methods include combining demineralized bone fragmentswith a carrier such that the tissue repair composition produced betweenabout 5 wt % and about 80 wt % or between about 30 wt % and about 50 wt% demineralized bone fragments. In some embodiments, the demineralizedbone fragments and the carrier may be combined with at least one of thecomponent, as described above. The methods may include packaging theinventive tissue repair compositions, in certain embodiments. In someembodiments, inventive methods of the present invention includeproviding at least one of bone tissue or bone tissue fragments and atleast one connective tissue, and preparing demineralized bone fragmentsand homogenized connective tissue from the at least one bone tissue andthe at least one connective tissue, as described above. Bone fragmentsmay include at least one of bone particles and bone fibers from bonetissue. Some methods of preparing the tissue repair compositions of thepresent invention may include the production of particles or fibers frombone tissue, as discussed above. Bone fragments may be demineralized, asdescribed above, in certain embodiments. The fragments may bedemineralized to have less than about 8 wt % residual calcium, less thanabout 4 wt % residual calcium, or between about 0.5 wt % and about 4 wt% residual calcium, in some methods of the present invention. Certainmethods of the present invention may include freeze-drying demineralizedbone fragments. In some embodiments, the demineralized bone fragmentsmay be freeze-dried to a point such that the freeze-dried fragments havean average residual moisture of less than about 10 wt %, or less thanabout 5 wt %. In some embodiments, freeze-dried demineralized bonefragments may be rehydrated before use in preparing the tissue repaircompositions of the present invention. Rehydrated freeze-drieddemineralized bone particles may have a residual moisture content ofless than about 80 wt %, less than about 50 wt %, less than about 25 wt%, or between about 25 wt % and about 10 wt %, in certain embodiments.

Certain methods for producing the inventive tissue repair compositionsmay include preparing a connective tissue or skin homogenate/carrier.Prior to homogenization, connective tissues (i.e., fascia, tendons,ligaments, pericardium, and articular cartilage, among others) or skinmay be crudely fragmented. Connective tissue (e.g., fresh orfreeze-dried) or skin may be sliced, ground, carved, chipped, chopped,minced, cut, dissected, rent, ripped, sectioned, snipped, diced, shaved,comminuted, or trimmed into crude fragments. In some embodiments, thecrude fragments may have an average diameter greater than about 50microns. The crude fragments may be of varying sizes, in someembodiments. Essentially intact connective tissue or crude fragments ofconnective tissue (e.g., fresh or freeze-dried) may be homogenized atleast one time to prepare the homogenate. Similarly, essentially intactskin or crude fragments of skin (e.g., fresh or freeze-dried) may behomogenized at least one time to prepare the homogenate. Thehomogenization step(s) of certain inventive methods may involveshear-induced shredding of connective tissue. Connective tissue or skinmay be homogenized to have tissue fragments having an average diameterof less than about 50 microns, less than about 20 microns, or less thanabout 50 microns and more than about 5 microns. Water, at least oneaqueous solution (e.g., isotonic saline) or other components may becombined with a connective tissue or skin before homogenization.

Certain methods include at least one of (a) heating a connective tissuebefore it is homogenized, (b) heating a connective tissue while it isbeing homogenized, and (c) heating a connective tissue homogenate. Insome embodiments the heating is done to a temperature of between aboutambient temperature and about 100° C., or between about 37° C. and about100° C. The heating may be carried out for between about 4 minutes andabout 30 minutes. The heating may be accomplished using sonication,microwave, irradiation, or conventional heat transfer from a heatingcomponent, among other methods known in the art.

In certain methods, the tissue repair composition may be cast in a mold.In some embodiments, a method may further include freeze-drying a castcomposition or cross-linking a cast composition utilizing chemicalreagents known in the art. Methods of the current invention may includesterilization of tissue repair compositions, components of tissue repaircompositions, and/or sterilization of packaged tissue repaircompositions/components. Sterilization may be performed using methodsknown in the art. The sterilization may involve the use of ionizingradiation, in some embodiments. In certain embodiments, the absorbeddose of ionizing radiation is between about 8.0 KGy and about 50 KGy,between about 8.0 KGy and about 25 KGy, and between about 8.0 KGy andabout 18 KGy. In some embodiments, the sterilizing step includes placingthe packaged composition on dry ice and irradiating the packagedcomposition. In certain embodiments, sterilization is performed at atemperature of between about −20° C. and −50° C. Certain methods of thepresent invention involve (a) providing at least one connective tissueand at least one bone tissue from at least one cadaver, (b)freeze-drying the connective tissue, (c) crudely fragmenting theconnective tissue, (d) adding at least one of water, an aqueous solution(i.e., isotonic saline) or a water miscible polar organic solvent to thecrude fragments to produce a mixture, which may optionally be heated,(e) homogenizing the mixture to produce a connective tissue homogenate,(f) fragmenting the bone tissue to produce fragments, (g) demineralizingthe bone fragments, (h) freeze-drying the demineralized bone fragments,(i) selecting demineralized bone fragments having sizes within aparticular range, (j) combining the selected demineralized bonefragments of the particular range with the connective tissue homogenate.Certain methods may include at least one of (a) heating a connectivetissue before it is homogenized, (b) heating a connective tissue whileit is being homogenized, (c) heating a connective tissue homogenate, and(d) heating the tissue repair composition. In some embodiments, heatingis sufficient to reach a temperature of about 100° C. A microwave ovenmay be used in the heating step, in certain embodiments. Connectivetissue homogenate may be heated and homogenized a second time beforebeing combined with the demineralized bone fragments in certain methodsof the present invention. In some embodiments, the selecting of bonefragments having sizes with a given range may involve the use of meshsieves. In some embodiments, the tissue repair composition may bepackaged, and the packaged composition may optionally be sterilized.

In certain embodiments, inventive tissue repair compositions of thepresent application may be applied to a prosthetic device utilized inneurological or orthopedic applications, to facilitate osteoconduction,and/or osteoinduction of native bone around the implant in order tobuild a stronger and more compatible association between the implant andthe native bone. Implantable bone prostheses may include a substrateformed of a biocompatible metal, ceramic, mineral component, orcomposite; and at least a partial coating of tissue repair composition.

Certain embodiments of the present invention are directed to prostheticdevices comprising, an implantable prosthetic device, and a coatingdirectly adjacent to at least a portion of a surface of the implantableprosthetic device. The coating includes at least one tissue repaircomposition comprising a plurality of demineralized bone fragments and ahomogenized connective tissue.

Some embodiments of the present invention are directed to a method ofcoating a prosthetic device comprising, providing an implantableprosthetic device, and applying at least one tissue repair compositionto at least a portion of a surface of the implantable prosthetic device.The tissue repair composition is as described above.

Further details of the process of the invention are presented in theexamples that follow:

EXAMPLES Example 1 Preparation of Tissue Repair Compositions ContainingFreeze-Dried Fascia

Fascia lata and bone from a human cadaver were procured and returned tothe processing facility under sterile conditions. Donor histories,personal and medical, were obtained following accepted standards of theAmerican Association of Tissue Banks. Microbiological tests wereperformed following FDA guidelines for testing sterility of products.

The bone and fascia were cleaned of unwanted tissues and freeze-dried.The freeze-dried fascia was cut into small (about ½ cm by ½ cm) pieces(e.g., crude fragments). Isotonic saline in a volume (1 cm³ of isotonicsaline corresponds to about 1 g) approximating 20 times the weight oftissue, was added to a container containing the cut fascia. Theingredients were heated to a temperature of 100° C. using a microwaveoven, and maintained at this temperature for 4 minutes. Water was addedto the heated composition to replace the liquid lost to evaporation. Theheated composition was transferred into a conventional blender andmechanically homogenized (e.g., blended) for 5 minutes. The homogenizedconnective tissue was re-heated for an additional 4 minutes in themicrowave oven, and mechanical homogenization was repeated for anadditional 5 minutes (e.g., until the mixture was liquefied andhomogeneous).

Ground demineralized bone powder was prepared by impact fragmentation ofbone, followed by freeze-drying. The freeze-dried particles were sizedusing mesh sieves. Ground demineralized bone particles having a size inthe range of about 250 to 710 microns and demineralized to an averageweight percent residual calcium of 2±1% were used.

To prepare a first tissue repair composition, the sized, freeze-drieddemineralized bone powder was added to the homogenized fascia until thefinal concentration of the bone was about 30% by weight. In a secondtissue repair composition, the demineralized bone powder was added tothe homogenized fascia until the final concentration of the bone wasabout 50% by weight. Samples of the tissue repair composition weresealed in sterilized glass vials in 2 g aliquots.

Example 2 Preparation of a Tissue Repair Composition ContainingFreeze-Dried Tendon

Tendon and bone from a human cadaver were procured and returned to theprocessing facility under sterile conditions. Donor histories, personaland medical, were obtained following accepted standards of the AmericanAssociation of Tissue Banks. Microbiological tests were performedfollowing FDA guidelines for testing sterility of products.

The bone and tendons were cleaned of unwanted tissues and freeze-dried.The freeze-dried tendon was cut into small (about cm by ½ cm) pieces(e.g., crude fragments). Isotonic saline in a volume (1 cm³ of isotonicsaline corresponds to about 1 g) approximating 20 times the weight oftissue, was added to a container containing the cut fascia. Theingredients were heated to a temperature of 100° C. using a microwaveoven, and maintained at this temperature for 4 minutes. Water was addedto the heated composition to replace the liquid lost to evaporation. Theheated composition was transferred into a conventional blender andmechanically homogenized (e.g., blended) for 5 minutes. The homogenizedconnective tissue was re-heated for an additional 4 minutes in themicrowave oven, and mechanical homogenization was repeated for anadditional 5 minutes (e.g., until the mixture was liquefied andhomogeneous).

Ground demineralized bone powder was prepared by impact fragmentation ofbone, followed by freeze-drying. The freeze-dried particles were sizedusing mesh sieves. Ground demineralized bone particles having a size inthe range of about 250 to 710 microns and demineralized to an averageweight percent residual calcium of 2±1 % were used.

In order to prepare the tissue repair composition, the freeze-drieddemineralized bone powder was added to the heated, homogenized tendontissue until the final concentration of the bone in the tissue repaircomposition was about 30% by weight. Samples were sealed in sterilizedglass vials in 2 g aliquots.

Example 3 Preparation of Tissue Repair Compositions ContainingNon-Freeze-Dried Fascia

Fascia and bone from a human cadaver were procured and returned to theprocessing facility under sterile conditions. Donor histories, personaland medical, were obtained following accepted standards of the AmericanAssociation of Tissue Banks. Microbiological tests were performedfollowing FDA guidelines for testing sterility of products.

The bone and fascia were cleaned of unwanted tissues and the bone wasfreeze-dried. Fresh, non-freeze-dried fascia was used. The fascia wascut into long strips and was mixed with water at a ratio of about 1:15by weight: The ingredients were heated to a temperature of 100° C. usinga microwave oven, and maintained at this temperature for 4 minutes.Water was added to the heated composition to replace the liquid lost toevaporation. The heated composition was transferred into a conventionalblender and mechanically homogenized (e.g., blended) for 5 minutes. Thehomogenized connective tissue was re-heated for an additional 4 minutesin the microwave oven, and mechanical homogenization was repeated for anadditional 5 minutes (e.g., until the mixture was liquefied andhomogeneous).

Ground demineralized bone powder was prepared by impact fragmentation ofbone, followed by freeze-drying. The freeze-dried particles were sizedusing mesh sieves. Ground demineralized bone particles having a size inthe range of about 250 to 710 microns and demineralized to an averageweight percent residual calcium of 2±1% were used.

To prepare a first tissue repair composition, the demineralized bonepowder was added to the homogenized, non-freeze-dried fascia until thefinal concentration of the bone was about 30% by weight. In a secondtissue repair composition, the demineralized bone powder was added to afinal concentration of about 50% by weight. Samples of the tissue repaircompositions were sealed in sterilized glass vials in 2 g aliquots.

Example 4 Preparation of a Tissue Repair Composition ContainingRehydrated Freeze-Dried Fascia and Rehydrated Freeze-Dried Bone

Fascia and bone from a human cadaver were procured and returned to theprocessing facility under sterile conditions. Donor histories, personaland medical, were obtained following accepted standards of the AmericanAssociation of Tissue Banks. Microbiological tests were performedfollowing FDA guidelines for testing sterility of products.

The bone and fascia were cleaned of unwanted tissues and freeze-dried.The fascia was rehydrated prior to being used in making of thecomposition. The rehydrated fascia was cut into small (about ½ cm by ½cm) pieces (e.g., crude fragments). Isotonic saline in a volume (1 cm³of isotonic saline corresponds to about 1 g) approximating 20 times theweight of tissue, was added to a container containing the cut fascia.The ingredients were heated to a temperature of 100° C. using amicrowave oven, and maintained at this temperature for 4 minutes. Waterwas added to the heated composition to replace the liquid lost toevaporation. The heated composition was transferred into a conventionalblender and mechanically homogenized (e.g., blended) for 5 minutes. Thehomogenized connective tissue was re-heated for an additional 4 minutesin the microwave oven, and mechanical homogenization was repeated for anadditional 5 minutes (e.g., until the mixture was liquefied andhomogeneous).

The ground demineralized bone powder was prepared by impactfragmentation, followed by freeze-drying, and finally the particles weresized using mesh sieves. Ground demineralized bone particles having asize in the range of about 125 to 500 microns were used.

The freeze-dried demineralized bone particles were rehydrated prior tobeing combined with the homogenized fascia. The final concentration ofthe rehydrated bone particles in the tissue repair composition was about20 wt %. The resulting tissue repair composition was a gel. The gel waspackaged in 5 mL Luerlock syringes. The gel was easily extruded from thesyringe. A thirteen-gauge needle was attached to the syringe, and thegel was easily extruded through the needle, as well.

Example 5 Preparation of a Molded Tissue Repair Composition ContainingFreeze-Dried Fascia

Fascia and bone from a human cadaver were procured and returned to theprocessing facility under sterile conditions. Donor histories, personaland medical, were obtained following accepted standards of the AmericanAssociation of Tissue Banks. Microbiological tests were performedfollowing FDA guidelines for testing sterility of products.

The bone and fascia were cleaned of unwanted tissues and freeze-dried.The freeze-dried fascia was cut into small (about ½ cm by ½ cm) pieces(e.g., crude fragments). Isotonic saline in a volume (1 cm³ of isotonicsaline corresponds to about 1 g) approximating 20 times the weight oftissue, was added to a container containing the cut fascia. Theingredients were heated to a temperature of 100° C. using a microwaveoven, and maintained at this temperature for 4 minutes. Water was addedto the heated composition to replace the liquid lost to evaporation. Theheated composition was transferred into a conventional blender andmechanically homogenized (e.g., blended) for 5 minutes. The homogenizedconnective tissue was re-heated for an additional 4 minutes in themicrowave oven, and mechanical homogenization was repeated for anadditional 5 minutes (e.g., until the mixture was liquefied andhomogeneous).

Ground demineralized bone powder was prepared by impact fragmentation ofbone, followed by freeze-drying. The freeze-dried particles were sizedusing mesh sieves. Ground demineralized bone particles having a size inthe range of about 125 to 710 microns and demineralized to an averageweight percent residual calcium of 2±1% were used.

In order to prepare the tissue repair composition, the freeze-drieddemineralized bone powder was added to the homogenized fascia until thefinal concentration of the bone in the composition was about 40% byweight. The tissue repair composition was then placed into differentcontainers and molds, and freeze-dried using a two-day cycle asprescribed by the manufacturer of the freeze-drier. The freeze-dried,molded, tissue repair compositions demonstrated high mechanical strengthand maintained the shape of their mold. The cast tissue repaircomposition may be rehydrated using an isotonic solution to make itmalleable or may be cross-linked with a fixative such as glutaraldehyde,EDC, or genapin to help retain its solid, rigid, molded form to be usedin applications where a specific shape and mechanical strength would bedesirable.

Example 6 Preparation of Tissue Repair Compositions ContainingFreeze-Dried Fascia

Fascia and bone from a human cadaver were procured and returned to theprocessing facility under sterile conditions. Donor histories, personaland medical, were obtained following accepted standards of the AmericanAssociation of Tissue Banks. Microbiological tests were performedfollowing FDA guidelines for testing sterility of products. The bone andfascia were cleaned of unwanted tissues and freeze-dried.

Ground demineralized bone powder was prepared by impact fragmentation ofbone, followed by freeze-drying. The freeze-dried particles were sizedusing mesh sieves. Ground demineralized bone particles having a size inthe range of about 125 to 500 microns and demineralized to an averageweight percent residual calcium of 2±1% were used.

The small pieces of connective tissue and the saline solution werebrought to a temperature of 100° C. using a heating plate and themixture was heated at this temperature for an additional 5 minutes.Water was added to the mixture to replace the solution lost due toevaporation. The mixture was transferred into a conventional blender andmechanically homogenized at approximately 15,000 rpm (maximum shearspeed of the commercially available blender) for 5 minutes. The mixturewas again heated to a temperature of 100° C. using the heating plate,and maintained at this temperature for an additional 5 minutes. Theheated mixture was again blended for two, 2-minute pulses to produce thehomogenized fascia.

To prepare a first tissue repair composition, the sized, freeze-drieddemineralized bone powder was added to the homogenized fascia until thefinal concentration of the bone was about 30% by weight. In a secondtissue repair composition, the demineralized bone powder was added tothe homogenized fascia until the final concentration of the bone wasabout 50% by weight.

Example 7 Preparation of Tissue Repair Compositions ContainingFreeze-Dried Fascia

Fascia and bone from a human cadaver were procured and returned to theprocessing facility under sterile conditions. Donor histories, personaland medical, were obtained following accepted standards of the AmericanAssociation of Tissue Banks. Microbiological tests were performedfollowing FDA guidelines for testing sterility of products.

The bone and fascia were cleaned of unwanted tissues and freeze-dried.The freeze-dried fascia was cut into small, ½ cm by ½ cm pieces (e.g.,crude fragments). Isotonic saline in a volume (1 cm³ of isotonic salinecorresponds to about 1 g) approximating 50 times the weight of thetissue to be processed was added to cut fascia. The fascia and salinewere brought to a temperature of 100° C. using a heating plate and washeated at this temperature for an additional 5 minutes. Water was addedto replace the solution lost due to evaporation. The heated material wastransferred into a blender and mechanically modified at 15,000 rpm(maximum shear speed of the commercially available blender) for 5minutes. The homogenized connective tissue was again heated to atemperature of 100° C. using the heating plate, and maintained at thistemperature for an additional 5 minutes. The heated homogenate wasblended for two, 2-minute pulses. The material was divided and placedinto centrifuge containers and spun at 1000 ref for 5, 7, 9, and 10minutes, respectively. Water in the material separated into a distinctlayer after the centrifugation process. The volume of the water layerwas proportional to the centrifugation time. The materials remainingafter removal of the water layer had different consistencies. To preparetissue repair compositions, the sized, freeze-dried demineralized bonepowder was added to the various homogenized materials until the finalconcentration of the bone was about 30% by weight. The viscosities ofthe tissue repair compositions correlated to the differing consistenciesof the homogenized connective tissue materials used in theirpreparation.

Example 8 Determination of New Bone Formation

Fascia lata and bone from a human cadaver were procured and returned tothe processing facility under sterile conditions. Donor histories,personal and medical, were obtained following accepted standards of theAmerican Association of Tissue Banks. Microbiological tests wereperformed following FDA guidelines for testing sterility of products.

The bone and fascia were cleaned of unwanted tissues and freeze-dried.The freeze-dried fascia was cut into small (about ½ cm by ½ cm) pieces(e.g., crude fragments). Isotonic saline in a volume (1 cm³ of isotonicsaline corresponds to about 1 g) approximating 20 times the weight oftissue, was added to a container containing the cut fascia. Theingredients were heated to a temperature of 100° C. using a microwaveoven, and maintained at this temperature for 4 minutes. Water was addedto the heated composition to replace the liquid lost to evaporation. Theheated composition was transferred into a conventional blender andmechanically homogenized (e.g., blended) for 5 minutes. The homogenizedconnective tissue was re-heated for an additional 4 minutes in themicrowave oven, and mechanical homogenization was repeated for anadditional 5 minutes (e.g., until the mixture was liquefied andhomogeneous).

Ground demineralized bone powder was prepared by impact fragmentation ofbone, followed by freeze-drying. The freeze-dried particles were sizedusing mesh sieves. Ground demineralized bone particles having a size inthe range of about 250 to 710 microns and demineralized to an averageweight percent residual calcium of 2±1% were used.

Tissue repair compositions having a putty-like consistency were preparedby adding the sized, freeze-dried demineralized bone powder to thehomogenized fascia until the final concentration of the bone was about24%, 26%, 30%, and 50% by weight, respectively.

The prepared tissue repair compositions, a demineralized bone matrix(DBM) control (without homogenized connective tissue), and a homogenizedtissue sample without DBM were implanted heterotopically (e.g., intomuscle pouches) in the hind quarters of athymic (e.g., nude) mice. Eachimplant (other than the homogenized tissue-only sample) contained 20 mgof demineralized bone matrix. The amounts of materials implanted werevaried to always implant 20 mg of DBM i.e., 40 mg of the 50% DBMcomposition was implanted. In that the DBM constituted 50% by weight,and the homogenized connective tissue constituted 50% by weight, thetotal implant of 40 mg contained 20 mg of DBM. Thus, 85 mg, 77 mg, 68mg, and 40 mg of the 24 wt %, 26 wt %, 30 wt %, and 50 wt % tissuerepair compositions were implanted, respectively. Three mice with twoimplants per mouse were used for each of the four tissue repaircompositions, the DBM control, and the homogenized tissue-only sample(e.g., 18 mice in all and 36 implants).

After 28 days, the implants were explanted, and one explant from eachmouse was fixed. At least one histological section was cut from thecenter of each of these explants. Samples were fixed in 10% bufferedformalin. Standard dehydration, embedding and sectioning protocols wereused to produce light microscopy slides that were-subsequently stainedwith hematoxylin and eosin. Using histomorphometric analysis, thepercent new bone formed was calculated as a cross-sectional area ofnewly formed bone (mm²) divided by the total cross-sectional area (mm²)for a representative microscopic view-of a histology slide multiplied by100. Every other field of view with at least 50% bone content was usedas a representative view with about 10 representative views beinganalyzed per slide.

The demineralized bone that was implanted without homogenized connectivetissue (e.g., the control) produced about 9.6% new bone growth, and thehomogenized fascia alone (freeze dried material) produced about 3.8% newbone growth. The 24%, 26%, 30%, 50% demineralized bone to homogenizedconnective tissue compositions resulted in about 6.4%, 11.1%, 14.5%, and16.2% new bone growth, respectively.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention. Other aspects, advantages, and modifications are within thescope of the following claims.

What is claimed is:
 1. A tissue repair composition comprising (i) one ormore bone component and (ii) connective tissue particles, wherein theconnective tissue particles have an average diameter greater than 5microns.
 2. The tissue repair composition of claim 1, wherein the bonecomponent is a demineralized bone fragment.
 3. The tissue repaircomposition of claim 2, wherein the composition comprises between 20%and 80% demineralized bone fragments.
 4. The tissue repair compositionof claim 2, wherein the tissue repair composition comprises a pluralityof demineralized bone fragments.
 5. The tissue repair composition ofclaim 4, wherein the demineralized bone fragments are demineralized bonefibers having an average thickness of between 0.1 mm and 0.3 mm and anaverage width of between 0.3 mm and 1.0 mm.
 6. The tissue repaircomposition of claim 1, wherein the composition is in the form of a gel,a paste, a putty, or a rehydratable free-dried form.
 7. The tissuerepair composition of claim 1, wherein the composition is in the form ofa gel.
 8. The tissue repair composition of claim 1, wherein thecomposition is injectable.
 9. The tissue repair composition of claim 8,wherein the composition is extrudable through a syringe having at leasta 13 gauge tube or needle.
 10. The tissue repair composition of claim 1,wherein the composition further comprises water, an aqueous solution, ora water miscible polar organic solvent.
 11. The tissue repaircomposition of claim 1, wherein the composition is aseptic or sterile.12. The tissue repair composition of claim 1, wherein the bone componentand/or the connective tissue particles is derived from allogenic orxenogenic sources.
 13. The tissue repair composition of claim 1, whereinthe bone component is cancellous, cortical, or cortico-cancellous bone.14. The tissue repair composition of claim 13, wherein the compositioncomprises bone components from more than one type of bone tissue. 15.The tissue repair composition of claim 14, wherein the bone component isa bone particle or a bone fiber.
 16. The tissue repair composition ofclaim 1, wherein the connective tissue particles are prepared from aconnective tissue obtained from a single source.
 17. The tissue repaircomposition of claim 1, wherein the connective tissue particles areprepared from a connective tissue from a single vertebrate source. 18.The tissue repair composition of claim 1, wherein the connective tissueparticles are derived from at least one of a human source, a bovinesource, an equine source, a porcine source, an ovine source, a caprinesource and a piscene source.
 19. The tissue repair composition of claim1, wherein the bone component is a demineralized, cortical boneparticle.
 20. The tissue repair composition of claim 1, wherein the bonecomponent is a demineralized, cancellous bone.
 21. The tissue repaircomposition of claim 1, wherein the connective tissue particles comprisecartilage tissue.
 22. The tissue repair composition of claim 21, whereinthe bone component is a demineralized bone.
 23. The tissue repaircomposition of claim 1, wherein the connective tissue particles have anaverage diameter of less than 50 microns.
 24. The tissue repaircomposition of claim 1, wherein the tissue repair composition comprisesthe bone component in an amount of between 5 wt % and 80 wt % of thetissue repair composition.
 25. The tissue repair composition of claim 1,wherein the tissue repair composition comprises the bone component in anamount of between 30 wt % and 50 wt % of the tissue repair composition.